The aims of this research are to identify the substances that serve as physiological regulators of hepatocyte proliferation, and to define the mechanisms through which they operate. Previous studies from our own and other laboratories, carried out mainly in whole animals, have implicated insulin, glucagon and epidermal growth factor (EGF). We have now turned to primary hepatocyte monolayer cultures for more definitive evaluation of these three substances, and to seek additional regulatory substances which we suspect may also be required. We have established a serum free system for short term culture of hepatic parenchymal cells in a chemically defined medium, in which contamination with other types of cells is negligible. We are beginning to test sera from normal and partially hepatectomized rats, and will attempt to define and isolate the active principles. We are also exploring effects of a variety of nutrients, hormones and known growth factors and their interactions and possible modes of operation.